Establish reliability of near-infrared spectroscopy (NIRS) mitochondrial capacity measurement and assess validity using heating and cooling interventions. We recruited 13 participants for four visits. Two visits consisted of a series of blood pressure occlusions proximal to the NIRS probe and electrical stimulation to the thigh, calf and forearm for reliability. Visits 3 and 4 required heating or cooling of the three muscles, using the protocol above. The between day reliability coefficient of variation (CV) in the forearm was 3% and time constant (TC) average was 41.9 ± 9.2 seconds (s). The within day CV in the forearm = 4.5%, the calf = 5.5% and the thigh = 4.6% (n = 13). Within day reliability for the cold condition in the forearm = 5.9%, calf = 4.3% and thigh = 4.4% and within day for the hot condition in the forearm = 6.4%, calf = 5.3% and thigh = 4.6%. The cold condition increased TC in the forearm, but decreased TC with heat (p < 0.05). The thigh decreased mitochondrial capacity with heat (p < 0.05), but cold was no different from control. These results suggest NIRS mitochondrial capacity has good within and between day reliability and temperature changes the mitochondrial capacity time constant.